The biochemical control mechanisms underlying the secretion of mediators of immediate hypersensitivity will be investigated using isolated normal rat mast cells. The central approach outlined in his proposal will be to better characterize the pharmacologic and immunologic modulation of cyclic nucleotide levels and the possible mechanisms of action of cyclic nucleotides in these cells. Cyclic nucleotide levels will be determined before, during and after mediator release induced by several well-characterized stimuli. The enzymes involved in cyclic nucleotide metabolism will be better characterized and the subcellular distribution of these enzymes and the cyclic nucleotides will be determined. Cyclic nucleotide dependent and independent protein kinases and phosphorylases will be characterized and attempts will be made to demonstrate changes in protein phosphorylation during induction of mediator secretion. In addition, the role of calcium in mediator release, the mechanism of phospholipid enhancement of release, and the mechanisms of termination of the release process will be investigated including particular attention to possible effects on cyclic nucleotide metabolism. The results will be important in helping to clarify the mechanisms by by which cyclic nucleotides and other regulatory molecules modulate mast cell secretion.